Lsm710 confocal microscope

At DIV 8 neurons were scraped with pipet tip and allowed for 2 days to
regenerate. Then, cells were fixed with 4% paraformaldehyde for 15 min, and then
permeabilized with 0.1% Triton X-100 in PBS for 15 min. Regenerating axons in
the scrape zone were visualized using an antibody against βIII tubulin
(1:2000, mouse monoclonal; catalog #G712A; Promega). FLAG-Rph3a was stained with
anti-FLAG (1:1000, Sigma-Aldrich, #F7425) antibody. Then, either Alexa-488 or
Alexa-568 conjugated donkey anti-rabbit IgG and Alexa-647 conjugated donkey
anti-mouse IgG (1:2000, all from Invitrogen) were used to detect primary
antibodies. Growth cones were visualized by staining for F-actin using
Alexa-488- or rhodamine-conjugated phalloidin (1:2000, catalog #A12379 or #R415,
Life Technologies). Samples were mounted with mounting solution (Vector
Laboratories) and observed using a LSM710 confocal microscope with 63×
objection lens.

At 36 hours after transfection, Cos7 cells expressing FLAG-NgR1 and HA-ORL1 were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 15 min. The samples were incubated overnight at 4°C with anti-FLAG (1:1000) and anti-HA (1:1000), or anti-NgR1 (1:1000), anti-HA (1:1000), and anti-GM130 (1:500) antibodies. Then, either Alexa 488–conjugated donkey anti-mouse immunoglobulin G (IgG) and Alexa 568–conjugated donkey anti-rabbit IgG, or Alexa 488–conjugated donkey anti-goat IgG, Alexa 568–conjugated donkey anti-rabbit IgG, and Alexa 647–conjugated donkey anti-mouse IgG (1:2000; all from Invitrogen) were used to detect primary antibodies. Samples were mounted with mounting solution (Vector Laboratories) and observed using an LSM710 confocal microscope.